Figure 2.

Structural consequences of H294 protonation.A, analysis by the hydrogen bond estimation algorithm DSSP of the secondary structure in the neutral (left) and the protonated holo-state (right). Legend: S, bend; T, hydrogen bonded turn; I, 5-helix; G, 3-helix; E, extended strand, part of β-ladder; B, isolated β-bridge; H, α helix; O, unassigned. B, C_α-root-mean-square fluctuation (RMSF). C, carboxyl carbon–Ca²⁺ distance histograms for E285 (upper graph), E293 (middle graph), and D308 (lower graph). D, structure of the Ca²⁺-binding site showing the distances plotted in C with dashed lines. E, histogram of the minimum distance between Ca²⁺ and the side-chain N atoms (N_δ and N_ε) of H294. Solid lines, mean of the histograms calculated for each simulation replica. Shaded area, 95% confidence interval of the mean obtained by bootstrapping (1000 samples). ll, long-loop; sl, short-loop.